Human Cytomegalovirus (HCMV) is a leading cause of congenital birth defects and causes substantial disease in various immunosuppressed populations, including transplant recipients and hematological cancer patients. Utilizing liquid chromatography-tandem mass spectrometry-based metabolomics we have found that HCMV infection induces a metabolic program that includes activating glycolysis, fatty biosynthesis and pyrimidine biosynthesis. Our results indicate that pharmaceutical or RNAi-mediated inhibition of pyrimidine biosynthesis attenuates HCMV replication. Further, our metabolomic analysis indicates that inhibition of pyrimidine biosynthesis substantially reduces the levels of UDP-sugar metabolites in HCMV-infected but not uninfected cells, indicating that these metabolic consequences are specific to HCMV infection. The UDP-sugars are the essential precursors of protein glycosylation and, accordingly, we find that pyrimidine biosynthetic inhibition attenuates the glycosylation of virion proteins. Importantly, we find that media supplementation with UDP-sugar metabolites restores virion protein glycosylation and rescues high-titer HCMV replication in the face of pyrimidine biosynthetic inhibition. Given its importance to infection, the mechanisms through which HCMV directs pyrimidine biosynthesis towards UDP-sugar production could present novel targets for therapeutic intervention. Therefore, our current goal is to elucidate how HCMV modulates pyrimidine and nucleotide-sugar metabolism to support viral infection. We will accomplish this goal through the pursuit of the following aims: (1) Determine how virally-induced pyrimidine biosynthesis contributes to HCMV infection; (2) Elucidate the cellular mechanisms through which HCMV activates pyrimidine and nucleotide-sugar metabolic flux; (3) Determine the viral factors responsible for HCMV-mediated pyrimidine and nucleotide-sugar metabolic activation. We expect our proposed research will identify key mechanisms through which HCMV drives pyrimidine precursors to support viral infection, an outcome that will provide novel therapeutic targets to treat HCMV infection.